Identification of potent and selective inhibitors of PKR via virtual screening and traditional design.

Protein Kinase RNA-activated (PKR) inhibition is thought to be relevant for immunology due to the potential to reduce macrophage and dendritic cell responses to bacteria and its signaling downstream of TNFα. PKR is also associated with neuroscience indications such as Alzheimer's disease due to its activation by the double stranded DNA (dsDNA) virus HSV1, a virus suggested to be important in the development of AD. Studies exploring the mechanistic role of PKR with existing tool molecules such as the tricyclic oxindole C16 are clouded by the poor selectivity profile of this ATP-competitive, Type I kinase inhibitor. Type II kinase leads such as the benzothiophene or pyrazolopyrimidine scaffolds from literature are equally poor in their selectivity profiles. As such, it became necessary to identify more potent and selective chemical matter to better understand PKR biology. A dual approach was taken. The first step of the strategy included virtual screening of the AbbVie compound collection. A combination of pharmacophore-based and GPU shape-based screening was pursued to identify selective chemical matter from promiscuous leads. The second step of the strategy followed traditional compound design. This step initiated from a literature lead with PKR cross reactivity. Combined, the two parallel efforts led to identification of more selective leads for investigation of PKR biology.

Hybrid balloon valvuloplasty for management of severe mitral stenosis in four symptomatic, adult dogs.

Four adult dogs weighing <10 kg presented for the evaluation of severe mitral valve stenosis with clinical signs. Owing to the size of the dogs, a hybrid surgical and interventional approach was utilized for balloon valvuloplasty. A left lateral thoracotomy was performed to allow direct entry through the left atrial wall. Transesophageal echocardiography was utilized for the entirety of the procedure in all dogs, and fluoroscopy was additionally used in two dogs. One dog had mild to moderate intra-operative bleeding from the left atrial wall during the procedure, but no other intra-operative complications were observed. No dogs developed a clinically relevant amount of worsened mitral regurgitation. Based on mitral leaflet mobility and transmitral flow profiles, there was perceived improvement in all four dogs. One dog died 6 h after extubation due to respiratory arrest. The remaining dogs survived to discharge and had resolution of clinical signs at home and discontinuation of heart failure medications. One dog died of an unknown cause at five months and another developed atrial fibrillation, and the owners elected to euthanize at ten months after the procedure. One dog continues to do well six months after the procedure as of the time of this writing. Hybrid balloon valvuloplasty can be a viable management option for small breed dogs with severe mitral stenosis exhibiting clinical signs, and both transesophageal echocardiography and fluoroscopy can be used intra-operatively to assist in successful procedural outcomes.

Pre-procedural femoral vessel ultrasound in dogs with patent ductus arteriosus: diameter, image quality and relationship with arterial catheterization.

INTRODUCTION: Objectives: Patent ductus arteriosus (PDA) in dogs is often treated via minimally invasive transvascular occlusion using femoral artery access. This study compared ultrasound-derived diameter and image quality of the right femoral artery (RFA) and vein (RFV) in dogs with PDA using a linear ultrasound probe (L-P) and phased-array transthoracic echocardiography probe (TTE-P). The case outcome was assessed. ANIMALS, MATERIALS & METHODS: Forty-five client-owned dogs with PDA were prospectively enrolled. Ultrasound-measured RFA and RFV diameters were obtained on images acquired with both probes pre-operatively and compared using Bland-Altman plots. The image quality of RFA and RFV was scored on L-P and TTE-P images. RESULTS: Comparison of RFA and RFV diameter from L-P versus TTE-P images revealed: [Mean difference (limits of agreement): RFA = 0.009 mm (-0.78-0.79 mm), RFV = 0.523 mm (-1.75-2.79 mm)]. Image quality scores were significantly higher for L-P than TTE-P (P < 0.0001). In six small dogs, measurable images were unattainable with TTE-P. Dogs of similar body weight had variable RFA diameters. Twenty-seven dogs had RFA catheterization. In 21/27 dogs, RFA diameter exceeded the external diameter of the introducer used for catheterization, and in 6/27, it was smaller. CONCLUSIONS: Pre-procedural ultrasound of the RFA in dogs with PDA is useful given variable RFA diameter relative to body weight. Despite poorer image quality, RFA diameters from TTE-P images were very similar to L-P images on average, suggesting TTE-Ps are suitable for pre-procedural planning in most dogs. Vasospasm, hypotension or differences in the location of ultrasound measurement versus catheterization might produce variation in pre-procedural versus intraoperative RFA size.

Prediction of clinically important acquired cardiac disease without an echocardiogram in large breed dogs using a combination of clinical, radiographic and electrocardiographic variables.

INTRODUCTION: Large breed (LB) dogs develop dilated cardiomyopathy (DCM) and myxomatous mitral valve disease (MMVD). Echocardiography is required for a definitive diagnosis but is not always available. Our objective was to assess the clinical utility of thoracic radiographs alone and in combination with physical examination and electrocardiography findings for the prediction of clinically important DCM or MMVD in LB dogs. ANIMALS: Four hundred fifty-five client-owned dogs ≥20 kg with concurrent thoracic radiographs and echocardiogram. MATERIALS AND METHODS: Medical records were reviewed and stored thoracic radiographs and echocardiographic images were measured to classify dogs as normal heart size (NHS), preclinical DCM, clinical DCM, preclinical MMVD (with cardiomegaly), clinical MMVD, or equivocal. Dogs with preclinical MMVD, without cardiomegaly, were classified as NHS. Vertebral heart size (VHS) and vertebral left atrial size (VLAS) were measured. Receiver operating characteristic curves and prediction models were derived. RESULTS: Prevalence of MMVD (39.3%) was higher than the prevalence of DCM (24.8%), though most MMVD dogs (67.0%) lacked cardiomegaly and were classified as NHS for analysis. The area under the curve for VHS to discriminate between NHS and clinical DCM/MMVD or preclinical DCM/MMVD was 0.861 and 0.712, respectively, while for VLAS, it was 0.891 and 0.722, respectively. Predictive models incorporating physical examination and electrocardiography findings in addition to VHS/VLAS increased area under the curve to 0.978 (NHS vs. clinical DCM/MMVD) and 0.829 (NHS vs. preclinical DCM/MMVD). CONCLUSIONS: Thoracic radiographs were useful for predicting clinically important DCM or MMVD in LB dogs, with improved discriminatory ability when physical examination abnormalities and arrhythmias were accounted for.

Artificial cardiac pacemaker placement in dogs with a cohort of myocarditis suspects and association of ultrasensitive cardiac troponin I with survival.

INTRODUCTION: Artificial cardiac pacemakers (APs) are a common treatment for symptomatic bradyarrhythmias in dogs, some of which may be triggered by underlying myocarditis. Severely elevated cardiac troponin I (cTnI) concentrations support a diagnosis of myocarditis. The association of ultrasensitive-cTnI (US-cTnI) concentration with survival in a large cohort of dogs receiving APs is not described. ANIMALS, MATERIALS, AND METHODS: The study included 110 dogs receiving APs over a 5-year period. Medical records were retrospectively reviewed to characterize the entire population receiving APs, with further analysis in dogs with preprocedural US-cTnI concentrations (n = 64) classified as normal/group 1 (n = 11), mildly to moderately elevated/group 2 (n = 27), and severely elevated/myocarditis suspects/group 3 (n = 26). RESULTS: Median survival time was 1079 days for the entire population, 1167 days for group 2, 949 days for group 3, and not met in group 1. There was not a statistically significant difference in survival between group 2 and group 3. Overall, US-cTnI had a mild, negative association with survival. Age had a stronger negative association. Infectious etiologies were identified in a minority of group 3 cases. A possible association between severely elevated US-cTnI and a sudden death outcome was noted. CONCLUSIONS: The negative association of US-cTnI with survival outcomes was mild, with age having a larger effect. Although a sudden death outcome may be seen more commonly in myocarditis suspects, group 3 survival time was similar to that of the entire canine population. Plausible infectious causes of myocarditis were infrequently identified.

Improving public mental health services for trauma victims in South Carolina.

Studies have shown that trauma and posttraumatic stress disorder are highly prevalent among persons with serious mental illness who are treated in state-funded mental health systems. Nevertheless, there is strong evidence that many of these persons receive inadequate mental health services. South Carolina recently became one of at least 15 states whose departments of mental health have initiated efforts to better address these needs. The goals of this initiative are to sensitize stakeholders, influence policies, educate and train clinicians, and increase knowledge by supporting a strong empirical research platform. Current progress and future directions are described in this article.

The cognitive dismantling of Eye Movement Desensitization and Reprocessing (EMDR) treatment of Posttraumatic Stress Disorder (PTSD).

Twenty-seven subjects were exposed to standard Eye Movement Desensitization and Reprocessing (EMDR) treatment or a similar treatment without the explicit cognitive elements found in EMDR. Standardized psychometric assessments were administered (Structured Interview for Post Traumatic Stress Disorder, Impact of Event Scale, Revised Symptom Checklist-90) by independent assessors at pretest, posttest and two separate follow-up periods. Potential subjects met specific inclusion/exclusion criteria. Subjective measures including Subjective Units of Disturbance and Validity of Cognition assessments were also conducted. A two-factor repeated measures analysis of variance revealed that both treatments produced significant symptom reductions and were comparable on all dependent measures across assessment phases. The present findings are discussed in light of previous dismantling research that converges to suggest that several elements in the EMDR protocol may be superfluous in terms of the contribution to treatment outcome. These same elements have nevertheless entered unparsimoniously into consideration as possible explanatory variables.

15-substituted lanosterols: post-transcriptional suppressors of 3-hydroxy-3-methylglutaryl coenzyme A reductase.

The oxolanosterol oxime 3 beta-hydroxylanost-7-en-15-one 15-oxime and the structurally similar 3 beta-hydroxylanost-7-en-15-one are dual-action inhibitors of cholesterol synthesis which cause both inhibition of lanosterol 14 alpha-methyl demethylase and suppression of the rate-limiting enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR). This report examines the mechanism by which these compounds lower HMGR protein levels in Chinese hamster ovary fibroblasts. Data are presented which suggest that both sterols reduce the translational efficiency of the HMGR mRNA as well as increase the rate of enzyme degradation. The effect of these sterols on the concentration of the low-density lipoprotein receptor (LDLR) in normal human fibroblasts was also determined. In these cells, both lanosterol analogs lowered HMGR protein levels without affecting LDLR concentration. This in contrast to the previously reported coordinate transcriptional regulation of these two genes by the C27-sterol 25-hydroxy-cholesterol. These findings are consistent with the hypothesis that different sterols regulate HMGR activity through distinct mechanisms.

Oxolanosterol oximes: dual-action inhibitors of cholesterol biosynthesis.

A series of oxolanosterol oximes and oxime ethers have been prepared as potential dual-action inhibitors of cholesterol biosynthesis. The synthesis of these oximes along with the evaluation of their ability to inhibit lanosterol 14 alpha-methyl demethylase (P450DM) and to suppress 3-hydroxy-3-methylglutaryl coenzyme. A reductase (HMGR) activity is presented. 3 beta-Hydroxylanost-7-en-15-one 15-oxime XIX was found to be an effective inhibitor of P450DM in rat liver microsomal preparations. In [14C]acetate incorporation studies using Chinese hamster ovary (CHO) cells, compound XIX was found to cause a dramatic reduction in the incorporation of acetate into C27 sterols with a concomitant increase in radiolabeled C30 sterols which is consistent with the inhibition of P450DM. In addition, 15-oxime XIX was shown to suppress HMGR activity in both wild-type CHO and P450DM-deficient (AR45) cells, indicating that suppression of HMGR is independent of any effects of this oxime on P450DM. In both cell lines, parallel declines in HMGR activity and HMGR protein levels were observed suggesting that compound XIX suppresses HMGR activity by regulation of gene expression. These results demonstrate that, as predicted, 15-oxime XIX is indeed a dual-action inhibitor of cholesterol biosynthesis which causes both the inhibition of P450DM and a reduction in HMGR activity.

32-Methyl-32-oxylanosterols: dual-action inhibitors of cholesterol biosynthesis.

Lanosterol 14 alpha-methyl demethylase (P-450DM) is the cytochrome P-450 monooxygenase which oxidatively removes the 14 alpha-methyl group of lanosterol. This demethylation is considered to be a rate-limiting step in the conversion of lanosterol to cholesterol. The intermediates in this transformation are known to bind very tightly to P-450DM and have been implicated in the regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) activity, the rate-limiting enzyme in overall cholesterol biosynthesis. Three 32-methylated analogs of the intermediates generated during the removal of the 14 alpha-methyl group by P-450DM, compounds 17a, 17b, and 18, have been prepared and their biochemical activities assessed. All three compounds were found to be direct inhibitors of P-450DM. These compounds were also shown to suppress HMGR activity by reducing the level of enzyme protein.